Abstract 1058: Novel Regulation of Wnt-Inducible Secreted Protein 1 by TNF-alpha in Primary Human Cardiac Fibroblasts

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
James T Colston ◽  
Sam D de la Rosa ◽  
Steven R Bailey ◽  
Bysani Chandrasekar
Keyword(s):  
Dna Binding ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Secreted Protein ◽  
Dependent Manner ◽  
Potent Inducer ◽  
Reporter Activity ◽  
Creb Activation ◽  
Human Cardiac Fibroblasts

Wnt1-induced secreted protein-1 (WISP-1) is a member of the CCN family of growth factors known to play a role in cellular growth, transformation, and survival. We previously demonstrated that WISP-1 is upregulated in the heart post-infarct, stimulates fibroblast proliferation and collagen expression, and is induced by the proinflammatory cytokine TNF-a. The present study was designed to investigate the molecular mechanisms involved in TNF-a-mediated WISP-1 induction in primary human cardiac fibroblasts (hCF). hCF were treated with rhTNF-a. WISP-1 expression was analyzed by Northern, Western and promoter-reporter assays. ERK1/2 and JNK activations by Western blotting using activation-specific antibodies and imunecomplex kinase assays. CREB activation by Western blotting, EMSA and reporter assay. Results demonstrate that TNF-a potently induces WISP-1 mRNA and protein expression in a time- and dose-dependent manner, effects that were blocked by anti-TNFR2, but not anti-TNFR1, neutralizing antibodies. Further investigations revealed that TNF-a stimulates WISP-1 promoter-reporter activity, an effect that was blunted when the core CREB DNA binding sequence was mutated or following the overexpression of dnCREB. TNF-a treatment induced CREB phosphorylation, in vitro and in vivo DNA binding, and reporter gene activities. TNF-a also induced ERK1/2 phosphorylation and activity, effects that were blocked by the MEK1 inhibitor U0126 and the ERK1/2 inhibitor PD98059. Furthermore, inhibition of ERK1/2 blunted CREB activation while inhibition of ERK1/2 and overexpression of dnCREB attenuated TNF-a-mediated WISP-1 promoter-reporter activity and mRNA expression. However, inhibition of JNK by SP600125 failed to modulate TNF-a-mediated WISP-1 induction. Most importantly, siRNA-mediated WISP-1 knockdown attenuated TNF-a-mediated hCF proliferation. Thus, our results demonstrate for the first time that TNF-a is a potent inducer of WISP1 expression in hCF, and TNF-a induces WISP-1 expression via TNFR2, MEK1, ERK, and CREB. Since TNF-a and WISP-1 are upregulated post-infarct, our results indicate that TNF-a/WISP-1 signaling may play a critical role in post-infarct myocardial remodeling.

IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-κB, and AP-1 activation

2007 ◽  
Vol 293 (6) ◽  
pp. H3356-H3365 ◽  
Author(s):  
Dolores M. Cortez ◽  
Marc D. Feldman ◽  
Srinivas Mummidi ◽  
Anthony J. Valente ◽  
Bjorn Steffensen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
P38 Mapk ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Dominant Negative ◽  
Expression Vectors ◽  
Type I ◽  
Mrna Levels ◽  
Human Cardiac Fibroblasts

Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways involved. HCF were treated with recombinant human IL-17. MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-α, or IL-1β antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-κB (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-β DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-κB, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-κB, and C/EBP-β activation and suggest that IL-17 may play a critical role in myocardial remodeling.

Elucidation of the Role of LMO2 (LIM-only protein 2) in Erythroid Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3443-3443
Author(s):  
AI Inoue ◽  
Tohru Fujiwara ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cord Blood ◽  
Western Blotting ◽  
Target Genes ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract Abstract 3443 Background: Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods: For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results: siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus. We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion: Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures: No relevant conflicts of interest to declare.

scholarly journals P0975EFFECT OF REGULATED INTRAMEMBRANE PROTEOLYSIS ON MEGALIN EXPRESSION DURING OXIDATIVE STRESS EXPOSURE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Yoshifumi Kurosaki ◽  
Futoshi Ikarashi ◽  
Akemi Imoto ◽  
Fumitaka Kawakami ◽  
Masanori Yokoba ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Western Blotting ◽  
Critical Role ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Gamma Secretase ◽  
Sprague Dawley ◽  
Intramembrane Proteolysis ◽  
Regulated Intramembrane Proteolysis

Abstract Background and Aims Megalin, an endocytic receptor in proximal tubular cells, plays a critical role in renal tubular protein reabsorption. We previously reported that oxidative stress induced the temporally increase in renal megalin expression through the PI3K/AKT signaling pathway, but that megalin elevation is normalized or decreased during long term exposure to oxidative stress (hydrogen peroxide). However, the underlying mechanisms are unclear. Studies have addressed that megalin is subjected to regulated intramembrane proteolysis (RIP). Intracellular megalin COOH-terminal fragment (MCTF) is produced by protein kinase C-regulated, metalloprotease-mediated ectodomain shedding and further cleavage by gamma-secretase to produce the soluble megalin intracellular domain (MICD). The MICD in turn translocates to the nucleus where it decreases expression of the Lrp2 gene encoding megalin. In the present study, we evaluated the effect of megalin RIP on the oxidative stress-regulated megalin expression. Method HK-2 cells were cultured with hydrogen peroxide (0.4 mmol/l) for 4.5 or 24 h, followed by treatment with gamma-secretase inhibitor, Compound E (5 mmol/L) or PKC activator, Phorbol 12-myristate 13-acetate (PMA, 0.5 mmol/L). Megalin expression was determined by performing western blotting or real-time PCR. The MCTF in medium was detected by western blotting. In animal experiments, Sprague-Dawley rats were randomly divided into two groups (n = 5): (i) STZ group (diabetic phenotype induced by streptozotocin administration) and (ii) sham group (vehicle). Urine was collected at two weeks after STZ administration, and the excretion of MCTF in urine was analyzed. Results Treatment of HK-2 cells with hydrogen peroxide (0.4 mmol/L) significantly increased megalin protein and mRNA levels at 4.5 h. Pretreatment of Compound E showed further increase in megalin expression in hydrogen peroxide-exposed cells. It was also found that presenilin-1 and -2, which are components of gamma-secretase, double knockdown with siRNA increased megalin expression in hydrogen peroxide treated-cells. On the other hand, PMA treatment inhibited the increase in both megalin protein and mRNA levels. In the cells treated with hydrogen peroxide for 24 h, megalin mRNA levels were normalized, but pretreatment of Compound E kept the elevation in megalin mRNA levels at 24 h after the treatment with hydrogen peroxide. Interestingly, megalin MCTF in the medium was increased by hydrogen peroxide treatment in a dose-dependent manner. Furthermore, megalin MCTF excretion in the urine of STZ-induced diabetes was significantly increased compared to sham rats. Conclusion These results suggested that oxidative stress-induced megalin upregulation was inhibited by RIP activation of megalin, suggesting that megalin RIP plays a role as a negative feedback system to oxidative stress-induced megalin upregulation. Furthermore, our data indicate that oxidative stress induces urinary excretions of MCTF in diabetic rats during the normoalbuminuric stage and potentially act as a marker of diabetic kidney disease.

Abstract 69: Angiotensin II- Canonical TGF-β Signaling Downregulates Apelinergic Pathway in Hypertension

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Rahul Rai ◽  
Varun Nagpal ◽  
Amanda E Boe ◽  
Douglas E Vaughan
Keyword(s):  
Blood Pressure ◽  
Angiotensin Ii ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Aortic Endothelial Cells ◽  
Nuclear Signaling ◽  
Human Cardiac Fibroblasts ◽  
Human Aortic Endothelial Cells

Background: Apelin is a novel peptide which along with its receptor, APJ, mediates apelinergic signaling. Apelinergic signaling plays a critical role in cardiovascular homeostasis, including regulation of blood pressure. While exogenous apelin blocks Angiotensin II (AngII) mediated nuclear signaling, the role and regulation of endogenous apelin in hypertension (HTN) remains obscure. We hypothesize that apelinergic pathway is downregulated in HTN, which is primarily mediated by aberrant AngII signaling. Approach: To test our hypothesis we utilized two mouse models of HTN, including AngII infusion and oral administration of N (ω)-nitro-l-arginine methyl ester (L-NAME). Blood pressure was monitored via noninvasive tail-cuff device. To determine the signaling involved we investigated the effect of AngII on apelinergic pathway in vitro. Results: Cardiac apelin was decreased significantly in both murine models of HTN. Downregulated apelin also corresponded with increased deposition of collagen, and up-regulation of senescence markers including PAI-1. Meanwhile, APJ levels were unaffected in both these hypertensive models. In our in vitro studies AngII downregulated apelin expression in human aortic endothelial cells (HAECs) and human cardiac fibroblasts (HCFs). Furthermore, our studies in AngII infused mice and in HCFs highlight the role of TGF-β-pSMAD signaling, independent of MEK involvement, in AngII induced apelin downregulation. Conclusion and Significance: Our studies demonstrate that aberrant AngII signaling downregulates apelin in HTN. This downregulation involves canonical Tgf-β1 signaling and affects apelin transcription. Importantly, we propose that AngII mediates its hypertensive pathology by decreasing apelinergic regulation. Since exogenous apelin blocks AngII signaling, further knowledge and negation of AngII induced apelin downregulation could result in the development of novel anti-hypertensive therapies.

scholarly journals Activation in M1 but not M2 Macrophages Contributes to Cardiac Remodeling after Myocardial Infarction in Rats: a Critical Role of the Calcium Sensing Receptor/NRLP3 Inflammasome

2015 ◽  
Vol 35 (6) ◽  
pp. 2483-2500 ◽  
Author(s):  
Wenxiu Liu ◽  
Xin Zhang ◽  
Meng Zhao ◽  
Xiaohui Zhang ◽  
Jinyu Chi ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Western Blotting ◽  
Nlrp3 Inflammasome ◽  
Cardiac Remodeling ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Inflammasome Activation ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Collagen Secretion

Aims: Macrophage (MΦ) infiltration during myocardial infarction (MI) amplifies cardiac inflammation and remodeling. We investigated whether activation of the NRLP3 inflammasome by a calcium sensing receptor (CaSR) in MΦ subsets contributes to cardiac remodeling following MI. Methods and Results: Infiltrated MΦ exhibited biphasic activation after MI; M1MΦ peaked at MI 3d and decreased until MI 14d, whereas M2MΦ peaked at MI 7d and decreased at MI 14d as shown via immunohistochemistry. IL-1β co-infiltrated with both M1MΦ and M2MΦ; IL-1β exhibited the same infiltrating tendency as M1MΦ, which was detected by immunohistochemistry. Increasing ventricular fibrosis was confirmed by Masson staining. CaSR and NLRP3 inflammasome in the MI group were upregulated in MΦ subsets in myocardium and peritoneal MΦ (p-MΦ) compared with the sham groups which were detected by immunofluorescence and western blotting. CaSR-activated NLRP3 inflammasome played a role in M1MΦ via PLC-IP3 but did not play a role in M2MΦ which were polarized by the THP-1 as shown by western blotting and intracellular calcium measurement. CaSR/NLRP3 inflammasome activation in M1MΦ led to the following effects: upregulated α-sma, MMP-2 and MMP-9, and collagen secretion; and downregulated TIMP-2 in cardiac fibroblasts via IL-1β-IL-1RI, which was detected by coculturing M1MΦ and cardiac fibroblasts. Conclusions: We suggest that the CaSR/NLRP3 inflammasome plays an essential role via the PLC-IP3 pathway in M1MΦ to promote cardiac remodeling post-MI in rats, including accelerated cardiac fibroblast phenotypic transversion, increased collagen and extracellular matrix (ECM) secretion; however, the CaSR/NLRP3 inflammasome does not play a role in this process in M2MΦ.

scholarly journals Investigating the potential for reversal of myofibroblast activation in human cardiac fibroblasts in 2D culture

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
C Hall ◽  
JP Law ◽  
JS Reyat ◽  
L Fabritz ◽  
P Kirchhof ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Protein Expression ◽  
Young’S Modulus ◽  
Western Blotting ◽  
Young's Modulus ◽  
Cardiac Fibroblasts ◽  
Public Institution ◽  
Young’S Moduli ◽  
Human Cardiac Fibroblasts

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): BHF MRC Introduction Cardiac fibroblasts (cFbs) are responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signalling processes. Prolonged and excessive activation of cFbs, via stimulation by transforming growth factor β (TGF-β), causes conversion of cFbs into myofibroblasts. Myofibroblasts are believed to cause pathological cardiac remodelling and to contribute to heart failure and arrhythmias. Reversion of myofibroblasts into cFbs has been demonstrated in rodent cells; it has yet to be explored in human cells. Purpose To characterise the effects of long-term 2D standard culture on the activation status of human cFbs. To identify the potential for human myofibroblasts to dedifferentiate back to cFbs. Methods Primary human cFbs were cultured in Corning Costar flasks (Young’s modulus E = ∼3GPa) for up to 10 passages. Cells were subsequently plated onto dishes with a Young’s modulus of ∼3GPa, 25kPa and 2kPa in the presence or absence of TGF-β (10ng/ml) and/or TGF-β receptor I inhibitor SD208 (10nM) for up to 4 days. The proliferative capacity of the cells was assessed using the CyQUANT NF® assay. Cells were assessed for mRNA and protein expression of myofibroblast activation markers α-smooth muscle actin (α-SMA) and collagen-1 by qPCR and western blotting. The localised distribution of α-SMA was assessed by confocal microscopy. Results Human cardiac fibroblasts robustly expressed α-SMA. Proliferation was significantly decreased at 2kPa compared to higher Young’s moduli (mean percentage change over 2 days: 2kPa = 115.1, 25kPa = 191.4, 3GPa = 205.9, p < 0.0001). qPCR analysis revealed no significant changes in expression of myofibroblast gene markers α-SMA and collagen 1 at either ∼3GPa, 25kPa or 2kPa Young’s Moduli in the presence or absence of TGF-β treatment (median fold change (interquartile range [IQR]) versus control: TGF-β(α-SMA, 3GPa) = 1.226 (0.820); TGF-β(Collagen 1, 3GPa) = 1.636 (1.403); TGF-β(α-SMA, 25kPa) = 1.069 (7.030); TGF-β(Collagen 1, 25kPa) = 1.103 (0.411); TGF-β(α-SMA, 2kPa) = 0.800 (5.021); TGF-β(Collagen 1, 2kPa) = 1.629 (7.092); n = 2-3). These data was confirmed by western blotting (median relative protein expression (IQR) versus control: TGF-β(α-SMA, 3GPa) = 1.012 (0.500); TGF-β(Collagen 1, 3GPa) = 1.008 (1.466); TGF-β(α-SMA, 25kPa) = 1.321 (2.282); TGF-β(Collagen 1, 25kPa) = 0.944 (1.125); TGF-β(α-SMA, 2kPa) = 1.142 (0.705); TGF-β(Collagen 1, 2kPa) = 0.283 (1.127), p > 0.05; n = 2-3). TGF-β or SD208 treatment did not affect α-SMA expression when assessed by confocal microscopy. Conclusions Long-term culture of human cFbs in 2D format leads to a robust and persistent activation of myofibroblasts that is unresponsive to TGF-ß activation or inhibition. Ongoing work is focussed on investigating whether human myofibroblast de-differentiation is possible.

scholarly journals Chlorogenic Acid Inhibits BAFF Expression in Collagen-Induced Arthritis and Human Synoviocyte MH7A Cells by Modulating the Activation of the NF-κB Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaohong Fu ◽  
Xilin Lyu ◽  
Han Liu ◽  
Dan Zhong ◽  
Zhizhen Xu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chlorogenic Acid ◽  
Promoter Region ◽  
Critical Role ◽  
Binding Activity ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Dna Binding Activity ◽  
Dna Binding Site ◽  
Dose Dependent

B cell activating factor (BAFF), a member of the tumor necrosis factor (TNF) family, plays a critical role in the pathogenesis and progression of rheumatoid arthritis (RA). Chlorogenic acid (CGA) is a phenolic compound and exerts antiarthritic activities in arthritis. However, it is not clear whether the anti-inflammatory property of CGA is associated with the regulation of BAFF expression. In this study, we found that treatment of the collagen-induced arthritis (CIA) mice with CGA significantly attenuated arthritis progression and markedly inhibited BAFF production in serum as well as the production of serum TNF-α. Furthermore, CGA inhibits TNF-α-induced BAFF expression in a dose-dependent manner and apoptosis in MH7A cells. Mechanistically, we found the DNA-binding site for the transcription factor NF-κB in the BAFF promoter region is required for this regulation. Moreover, CGA reduces the DNA-binding activity of NF-κB to the BAFF promoter region and suppresses BAFF expression through the NF-κB pathway in TNF-α-stimulated MH7A cells. These results suggest that CGA may serve as a novel therapeutic agent for the treatment of RA by targeting BAFF.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

scholarly journals Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 286
Author(s):  
Mary Frances Nakamya ◽  
Moses B. Ayoola ◽  
Leslie A. Shack ◽  
Mirghani Mohamed ◽  
Edwin Swiatlo ◽  
...  
Keyword(s):  
Stress Responses ◽  
Critical Role ◽  
Acid Stress ◽  
Arginine Decarboxylase ◽  
Arginine Deiminase ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Opportunistic Human Pathogen ◽  
Thiol Peroxidase ◽  
The Impact

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

scholarly journals Colorectal Cancer Apoptosis Induced by Dietary δ-Valerobetaine Involves PINK1/Parkin Dependent-Mitophagy and SIRT3

2021 ◽  
Vol 22 (15) ◽  
pp. 8117
Author(s):  
Nunzia D’Onofrio ◽  
Elisa Martino ◽  
Luigi Mele ◽  
Antonino Colloca ◽  
Martina Maione ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Mitochondrial Dysfunction ◽  
Cancer Cells ◽  
Current Knowledge ◽  
Apoptotic Cell ◽  
Critical Role ◽  
Dependent Manner ◽  
Colon Cancer Cells ◽  
Protein Levels

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.

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